This disclosure relates to the use of phage display technology to identify peptides that bind to pathogenic fungi and more particularly to pathogenic fungi of the genera Phytophthora, Phakapsora, and Uromyces. Random peptide phage display libraries are constructed using degenerate oligonucleotides. Phage expressing the peptides on their surface are contacted with fungi at different life stages and those phage that bind are isolated, amplified and the peptides identified. Once identified, peptides can be screened for anti fungal activity and used to identify and characterize binding sites on fungi.
Phytophthora is an economically important disease causing organism in the United States causing large losses in many agronomically important crop species. Phytophthora sojae is the second most important pathogen of soybeans in the United States. (Doupnik, Plant Dis. 77:1170-1171, 1993). Phytophthora capsici has a broad host range and most notably limits production of high-value, solanaceous vegetable crops. Control of these pathogens is particularly difficult, often requiring treatment of entire fields with biocidal compounds. Although effective, increasing concern about the environmental and economic costs of such treatments require the need for alternative control methods.
Phytophthora species are obligate parasites adapted to long-term survival in soil in the absence of host plants. Oospores or chlamydospores exist in low densities in the soil and enable survival of the pathogen. In the presence of a susceptible plant, the pathogen progresses rapidly through a series of finely tuned developmental steps that produce cycles of infection and disease. Pathogen development from oospores or chlamydospores through zoospore release, encystment, germination and infection appear straight-forward at first glance. Yet, the procession of life stages is finely tuned to environmental signals, particularly those signals coming from a host plant.
Zoospores are the life-stage of greatest importance for dispersal to root infection sites. A major susceptible site is located just behind the apical meristem of the root where cells are elongating. Exudates released from elongating cells serve as signals that direct chemotactic movement of zoospores toward the site (Carlile, in Phytophthora: Its Biology, Taxonomy, Ecology and Pathology, Erwin et al., eds., APS Press, 1983; Deacon and Donaldson, Mycol. Res. 97:1153-1171, 1993). The zoospore chemotactic response varies with the composition of root exudates and is species specific. For example, zoospores of P. capsici, P. cactorum, and other species are attracted to an array of sugars and amino acids (Hickman, Phytopathology, 60:1128-1135, 1970; Khew and Zentmyer, Phytopathology, 63:1511-1517, 1973), but zoospores of P. sojae are attracted to specific isoflavonoid compounds (Norris et al., Plant Physiol., 117:1171-1178, 1998). Although the precise mechanism of chemoattraction is not known, Deacon and Donaldson (MycoL Res., 97:1153-1171, 1993) and Carlile (in Phytophthora: Its Biology, Taxonomy, Ecology and Pathology, Erwin et al., eds., APS Press, 1983) summarized experiments that suggested the involvement of chemoreceptors on the zoospore surface.
Zoospores encyst as they approach the root surface in response to environmental signals. Encystment of zoospores of P. palmivora and other Phytophthora species, for example, can be influenced by local calcium ion concentrations (Griffith et al., Arch. Microbiol., 149:565-571, 1988; Warburton and Deacon, Fungal Genetics Biol., 25:54-62, 1998). Encystment can also be induced by high concentration of chemoattractants or by root cell wall components. For example, zoospores of P. sojae encyst in the presence of high concentrations of soybean isoflavonoid compounds (Morris and Ward, Physiol. Mol. Plant Pathol., 40:17-22, 1992). In contrast, zoospores of Pythium aphanidermatum encysted when in contact with fucosyl and galactosyl residues from cell surfaces of cress roots (Longman and Callow, Physiol. Mol. Plant Pathol., 30:139-150, 1987; Estrada-Garcia et al., J Exp. Bot. 41:693-699, 1990). Deacon and Donaldson (Mycol. Res., 97:1153-1171, 1993) noted that encystment in the presence of high concentrations of attractants would be deleterious to infection potential, and thus selected against over time. They suggested, however, that attractants at the root surface may be sufficiently concentrated to predispose zoospores to encyst after contact with root surface residues.
When in contact with a root, zoospores encyst with a specific orientation so that a germ tube emerges toward the root. If zoospores encyst before contact with the root, the germ tubes will emerge in any orientation and must re-orient in order to locate the root and infect the plant. Cell surface receptors on the germ tube are thought to be involved in this root-orientation process. Morris et al. (Plant Physiol., 117:1171-1178, 1998), for example, demonstrated an oriented response of P. sojae germling growth to low, nontoxic concentrations of isoflavonoid compounds derived from soybeans. Zentmyer (Science, 133:1595-1596, 1961) reported hyphal orientation of P. cinnamomi toward host roots, but the nature of the attractant compound(s) was not defined.
After infection, hyphae grow through plant tissue intercelluarly and/or intracellularly depending on the species of pathogen (Stossel et al., Can. J Bot., 58:2594-2601, 1980; Coffey and Wilson, in Phytophthora: Its Biology, Taxonomy, Ecology and Pathology, Erwin et al., eds., APS Press, 1983; Enkerli et al., Can. J Bot., 75:1493-1508, 1997; Hardham and Mitchell, Fungal Gen. Biol., 24:252-284, 1998; Murdoch and Hardham, Protoplasma, 201:180-193, 1998). Haustoria are formed by some Phytophthora species, including P. infestans (Coffey and Wilson, in Phytophthora: Its Biology, Taxonomy, Ecology and Pathology, Erwin et al., eds., APS Press, 1983), P. capsici (Jones et al., Phytopathology, 64:1084-1090, 1974), and P. sojae (Stossel et al., Can. J Bot., 58:2594-2601, 1980). Both hyphae and haustoria establish close contact with host cell walls and membranes. Presumably, cell surface receptors are important in sensing plant signals, although direct evidence is lacking. Indirect evidence of cell surface receptors comes from observations such as the occurrence of vesicles in the distal portion of haustoria (Coffey and Wilson, in Phytophthora: Its Biology, Taxonomy, Ecology and Pathology, Erwin et al., eds., APS Press, 1983). Heath (Can. J Bot., 73(Suppl.):S 131 -S 139, 1995) discussed the possible events of fungal hyphal tip growth involving communication with the surrounding environment via ion channel and vesicle functions.
As discussed above, evidence points to the prominence of cell surface receptors in triggering behavioral and developmental steps of Phytophthora. Cells surface receptors, therefore, may provide a means for disrupting pathogen development and so infectivity. Delay or disruption of development can have a substantial impact, since zoospores have only a limited time to locate, contact, and penetrate an infection site that is effectively moving with the growing root tip. This time limitation results from the changing susceptibility of the root tissues, since as the tissues in the elongation region mature, they become significantly less susceptible to infection (English and Mitchell, Phytopathology, 78:1478-1483, 1988).
“Fusion phage” are filamentous bacteriophage vectors in which foreign peptides and proteins are cloned into a phage coat gene and displayed as part of a phage coat protein. The commonly used coat genes for the production of fusion phage are the pVIII gene and the pIII gene. About 3900 copies of pVIII make up the major portion of the tubular virion protein coat. Each pVIII coat protein lies at a shallow angle to the long axis of the virion, with its C-terminus buried in the interior close to the DNA and its N-terminus exposed to the external environment. Five copies of the pIII coat protein are located at the terminal end of each virion and are involved in attachment of the phage to pIII of E. coli and for virus reassembly after infection and replication. Peptides displayed as part of pVIII are constrained in the matrix of their display on the virion coat. In contrast, peptides displayed as part of pIII are more flexible due to the terminal position of the pIII proteins. Specific phage can be constructed to display peptides of six to 15 amino acids in length. Insertion of random or degenerate oligonucleotides into the coat protein genes allows the production of phage displayed random peptide libraries. A typical display library contains 10 to 100 copies of as many as 108 random sequence peptides. Thus, phage display is useful for screening for rare peptides with desired binding characteristics.
Phage-displayed random peptide libraries have been used for isolating ligands to cell surface receptors on mammalian cells. For example, peptides have been isolated from phage-displayed libraries that bind the transmembrane integrin glycoproteins involved in cell-extracellular matrix and cell-cell interactions (O'Neil et al, Proteins, 14:509-515, 1992; Smith et al., J. Biol. Chem., 269:32788-32795, 1994; Healy et al., Biochemistry, 34:3948-3955, 1995). The phage displayed peptides specifically blocked cell adhesion to defined extracellular molecules and other cells (Koivunen et al., J. Biol. Chem., 268:20205-20210, 1993; Koivunen et al., J Cell Biol., 124:373-380, 1994; Healy et al., Biochemistry, 34:3948-3955, 1995; Pasqualini et al., Nature Biotech., 15: 542-547, 1997). Phage-displayed random peptide libraries have also been used to select peptides that distinguish between brain and kidney tissue (Pasqualini and Ruoslahti, Nature, 380:364-366, 1996). In vivo, affinity-selection of phage-displayed random peptides has also been used to select peptides that bind selectively to endothelial cells of blood vessels of specific tumor tissues (Pasqualini et al., Nature Biotech., 15: 542-547, 1997). When these peptides were fused to an anti-cancer drug and injected into tumor-bearing mice, the peptides successfully targeted the drug to tumor blood vessels and deterred progressive tumor development (Arap et al., Science, 279:377-380, 1998).
Phage-display methods have been applied to plant pathogens in only very limited circumstances. Phage display methods have been used almost exclusively to identify antibodies for plant virus diagnosis (Susi et al., Phytopathology, 88:230-233, 1998; Ziegler et al., Phytopathology, 88:1302-1305, 1998; Griep et al., J Plant Pathol., 105:147-156 1999; Toth et al., Phytopathology, 89:1015-1021, 1999). Phage display was used in a single instance to select antibodies with affinity to surface-exposed epitopes on germlings and spores of Phytophthora infestans (Gough et al., J Immunol. Methods, 228:97-108, 1999). Isolated phage-displayed, single-chain variable fragment (Fv) antibody fragments were not assessed for their potential to influence spore or germling behavior. Antibodies were tested for their antifungal activity with sporangia, but were found to have no detectable antifungal activity.
Phakopsora pachyrhizi is a fungus that causes a rust disease of soybean (Glycines max). The pathogen has spread from Asia to all other soybean production regions in the world. P. pachyrhizi arrived in the US during the fall of 2004. At present, there is no known durable resistance available in any soybean varieties. Uromyces appendiculatus is a fungus that causes rust on bean (Phaseolus vulgaris). Breeders are working to identify genes in bean that can be manipulated for rust resistance. U.S. soybean producers have anticipated the arrival of Phakopsora pachyrhizi, the fungus that causes soybean rust, since its reported occurrence in Brazil. The arrival of P. pachyrhizi in the U.S. last fall ended the anticipation, and farmers must now respond to the potential annual occurrence of this new disease. Farmer concerns have been based on reports of losses ranging from 10 to 80% in other regions of the world when control measures were not successfully implemented.
Soybean producers have been concerned because no durable, natural resistance to rust has been discovered after testing more than 18,000 US soybean varieties, and the pathogen, P. pachyrhizi can potentially infect any cultivar produced. In anticipation of the arrival of the rust pathogen, a great deal of research has been conducted to identify effective fungicides, and emergency governmental clearance for application to soybean has been obtained. Traditional screening and breeding methods have identified no major resistance genes to the aforementioned pathogens, and particularly in the case of U appendiculatus and P. pachyrhizi. 
Fungicides will likely be the front-line of defense for many years until new resistance genes or other forms of resistance are identified.
Fungicides have not traditionally been used in most soybean production. Consequently, there is limited information concerning the costs of this disease management practice and its likely economic viability. These concerns have led to variable estimates of the acreage in Missouri and other states that may be shifted from soybean to alternative crops.
To protect soybean farmers and to ensure that production meets national needs, alternatives to fungicides must be developed as rapidly as possible. This disclosure addresses a new type of biotechnology-based rust resistance. The technology involves the development and deployment of defense peptides in transgenic plants, such as soybeans. The technology similarly impacts the important field bean (Phaseolus vulgaris) rust pathogen, Uromyces appendiculatus. 
As rust-inducing fungi, U appendiculatus and P. pachyrhizi belong to the order Uredinales, within the class Basidiomycetes. U. appendiculatus produces five spore stages on a single host plant. P. pachyrhizi reproduces predominantly by uredospores on a single host plant. Uredospores are responsible for rapid spread of the fungus. P. pachyrhizi can infect dozens of legume species, in addition to soybean.
Uredospores of U appendiculatus penetrate through foliar stomatal openings. P. pachyrhizi differs in that germinated uredospores penetrate directly through the leaf epidermal cell layer. Typically, a uredospore that lands on a leaf surface germinates to produce an infection pad (appressorium) that adheres to the surface. In both species, the appressorium produces a hyphal peg that penetrates the plant. After penetration, each fungus develops thread-like structures (hyphae) that grow inter-cellularly through leaf tissues. The hyphae enter host cells without killing them. There, they produce spherical structures (haustoria) that extract nutrients from the living leaf cells. Soon after infection each fungus forms uredia that produce additional spores.
Combinatorial phage-display libraries provide a vast array of random peptides from which to select ligands directed to proteins of interest (O'Neil et al., 1992). Phage-display peptide libraries are mixtures of filamentous phage clones, each of which displays a single foreign peptide sequence on the virion surface (Cwirla, 1990; Scott and Smith, 1990). The displayed peptide is physically linked with its coding DNA in the phage genome. Thus, the peptide can be easily and quickly identified and transferred to other vectors or display systems. Typical libraries contain 109 random peptide variants.
Random peptide libraries are useful for isolating ligands of importance to cell-surface molecules of mammalian cells. For example, peptides with affinity for transmembrane glycoproteins, integrins, have been isolated from libraries by biopanning against purified molecules (O.Neil et al., 1992; Smith et al., 1994; Healy et al., 1995). Some peptides were found to block cell adhesion to defined extracellular molecules and to other cells. Effectiveness of peptide binding and inhibition was specific to peptide sequence motif (Koivunen et al., 1993; Koivunen et al., 1994; Healy et al., 1995; Pasqualini et al., 1995), and the effectiveness of selections was specific to particular organ tissues (Pasqualini and Ruoslahti, 1996).
What is needed, therefore, is a rapid and efficient method for screening peptides for specific binding to plant pathogens. Once identified, the peptides can be further evaluated for their ability to prevent infection of plants by the pathogen, and suitable peptides can be applied directly to the plant, used to treat the soil or, alternatively, sequences encoding the peptides can be introduced in the plants to confer immunity or resistance against the pathogen. In this manner, economical and environmentally safe and effective methods of controlling plant pathogens can be developed.